This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The goal of this research is to determine the molecular mechanisms that regulate the biosynthesis and signaling activity of the Pseudomonas quinolone signal (PQS). Opportunistic pathogen Pseudomonas aeruginosa causes chronic lung infections which are the major causes of mortality in patients suffering from cystic fibrosis. Moreover, P. aeruginosa is one of the most common nosocomial pathogens that accounts for 10% of all hospital-acquired infections. The pathogenicity of Pseudomonas is controlled by a set of extracellular molecules collectively known as the quorum sensing signals, which activate the production of extracellular virulence factors. Because of the essential roles of quorum sensing in the pathogenicity of P. aeruginosa, proteins involved in quorum-sensing have been recognized as targets for new antibacterial therapeutic development. Although the importance of PQS in Pseudomonas virulence is well established, some key steps in PQS biosynthesis are not characterized. Nor are the functions of PQS in the regulation of virulence fully understood. Genetic studies using P. aeruginosa mutant strains, which are defective in PQS signaling, have identified that proteins encoded by the pqs gene operon are required. Specifically, PqsA, PqsB, PqsC and PqsD are involved in PQS synthesis;whereas PqsE, which is the product of the last gene of the operon, is required for the PQS-dependent activation of the downstream virulence factors. However, there is very limited research on the functions of these proteins at the molecular level. We have preliminary data that PqsD catalyzes the formation of DHQ, a unacylated quinolone secreted by P. aeruginosa, by the Claisen condensation of malonyl-ACP (or CoA) with anthraniloyl-CoA. The aims of this proposal are: 1) to determine the exact order and reactions catalyzed by PqsB, PqsC and PqsD in the synthesis of PQS;2) to determine the role(s) of PqsE in the PQS signaling network and Pseudomonas virulence. We propose to use integrated approaches to study the functions and structures of these proteins with biochemical analyses and X-ray crystallography. The findings of the proposed research will not only allow us to better understand the PQS quorum sensing system, but also provide novel insights on new targets for antibacterial discovery by the inhibition of virulence.